(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
(19) World Intellectual Property Organization
International Bureau
(43) International Publication Date
7 December 2000 (07.12.2000)
PCT
(10) International Publication Number
WO 00/73785 A2
(51) International Patent Classification 7 : G01N 33/00
(21) International Application Number. PCT/USOO/15413
(22) International Filing Date: 31 May 2000 (31 .05.2000)
(25) Filing Language: English
(26) Publication Language: English
(30) Priority Data:
09/324,443
2 June 1999 (02.06.1999) US
(71) Applicant: NOVA BIOMEDICAL CORPORATION
[US/US]; 200 Prospect Street, Waltham, MA 02254 (US).
(72) Inventors: WINARTA, Handani; 18 Hyacinth Drive,
Nashua, NH 03062 (US). CAI, Xiaohua; 19 McCuUoch
Street, Needham, MA 02494 (US). SETO, Fung; 31
Pratt Drive, Newton, MA 02465 (US). YOUNG, Chung,
Chang; 145 Buckskin Drive, Weston, MA 02193 (US).
(74) Agent: DELEAULT, Robert, R.; Mesmer Law Offices,
P.A., 41 Brook Street, Manchester, NH 03104 (US).
(81) Designated States (national): AE, AL, AM, AT, AU, AZ,
BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK,
DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL,
IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU,
LV, MA. MD, MG, MX, MN, MW, MX, NO, NZ, PL, PT,
RO, RU, SD, SE, SG, SI, SK, SL. TJ, TM, TR, TT, TZ, UA,
UG, UZ, VN, YU, ZA, ZW.
(84) Designated States (regional): ARIPO patent (GH, GM,
KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), Eurasian
patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
patent (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE,
IT, LU, MC, NL, PT, SE), OAPI patent (BF, BJ, CF, CG,
CI, CM, GA, GN, GW, ML, MR, NE, SN, TD. TG).
Published:
— Without international search report and to be republished
upon receipt of that report.
For two-letter codes and other abbreviations, refer to the "Guid-
ance Notes on Codes and Abbreviations " appearing at the begin-
ning of each regular issue of the PCT Gazette.
== (54) Title: DISPOSABLE SENSOR AND METHOD OF MAKING
00
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(57) Abstract: A disposable electrode strip for testing a fluid sample including a laminated strip with a first and second end, a
reference electrode embedded in the laminated strip proximate to the first end, at least two working electrodes embedded in the.
laminated strip proximate to the first end and the reference electrode, an open path for receiving a fluid sample beginning from the
first end and being sufficiently long to expose the reference electrode and the working electrodes to the fluid sample, and conductive
contacts located at the second end of the laminated strip. The laminated strip has a base layer with a conductive coating, a reagent
holding layer, a channel forming layer and a cover. One of the working electrodes contains a reagent substantially similar to the
reagent of the reference electrode and a second working electrode contains a reagent having an enzyme.
WO 00/73785
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DISPOSABLE SENSOR AND METHOD OF MAKING
BACKGROUND OF THE INVENTION
5
1. Field of the Invention
The present invention relates generally to electrochemical sensors that
can be used for the quantification of a specific component or analyte in a liquid
sample. Particularly, this invention relates to a new and improved
10 electrochemical sensor and to a new and improved method of fabricating
electrochemical sensors. More particularly, this invention relates to a disposable
electrochemical sensor that is inexpensive to manufacture. Even more
particularly, this invention relates to a disposable electrochemical sensor that
gives accurate readings in the presence of interferants and varying red blood
15 cells (hematocrit). Still even more particularly, this invention relates to disposable
electrochemical sensors which are used for performing electrochemical assays
for the accurate determination of analytes in physiological fluids.
2. Description of the Prior Art
20 Biosensors have been known for more than three decades. They are used
to determine concentrations of various analytes in fluids. Of particular interest is
the measurement of blood glucose. It is well known that the concentration of
blood glucose is extremely important for maintaining homeostasis. Products that
measure fluctuations in a person's blood sugar, or glucose levels have become
25 everyday necessities for many of the nation's millions of diabetics. Because this
disorder can cause dangerous anomalies in blood chemistry and is believed to be
a contributor to vision loss and kidney failure, most diabetics need to test
themselves periodically and adjust their glucose level accordingly, usually with
insulin injections. If the concentration of blood glucose is below the normal
30 range, patients can suffer from unconsciousness and lowered blood pressure
which may even result in death, if the blood glucose concentration is higher than
the normal range, the excess blood glucose can result in synthesis of fatty acids
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and cholesterol, and in diabetics, coma. Thus, the measurement of blood
glucose levels has become a daily necessity for diabetic individuals who control
their level of blood glucose by insulin therapy.
Patients who are insulin dependent are instructed by doctors to check their
5 blood-sugar levels as often as four times a day. To accommodate a normal life
style to the need of frequent monitoring of glucose levels, home blood glucose
testing was made available with the development of reagent strips for whole
blood testing.
One type of blood glucose biosensors is an enzyme electrode combined
10 with a mediator compound which shuttles electrons between the enzyme and the
electrode resulting in a measurable current signal when glucose is present. The
most commonly used mediators are potassium ferricyanide, ferrocene and its
derivatives, as well as other metal-complexes. Many sensors based on this
second type of electrode have been disclosed. Examples of this type of device
1 5 are disclosed in the following patents.
U.S. Patent No. 5,628,890 (1 997, Carter et al.) discloses an electrode
strip having an electrode support, a reference or counter electrode disposed on
the support, a working electrode spaced from the reference or counter electrode
on the support, a covering layer defining an enclosed space over the reference
20 and working electrodes and having an aperture for receiving a sample into the
enclosed space, and a plurality of mesh layers interposed in the enclosed space
between the covering layer and the support. The covering layer has a sample
application aperture spaced from the electrodes. The working electrode includes
an enzyme capable of catalyzing a reaction involving a substrate for the enzyme
25 and a mediator capable of transferring electrons between the enzyme-catalyzed
reaction and the working electrode.
This device proposes to reduce the effect of hematocrit on the sensor
readings. According to the disclosure, this results from the downstream spacing
of the reference electrode relative to the working electrode in combination with
30 the thin layer of the sample solution created by the mesh layers.
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U.S. Patent No. 5,708,247 (1998, McAleer et al.) discloses a disposable
glucose test strip having a substrate, a reference electrode, a working electrode,
and a means for making an electrical connection. The working electrode has a
conductive base layer and a coating layer disposed over the conductive base
5 layer. The coating layer is a filler having both hydrophobic and hydrophilic
surface regions which form a network, an enzyme and a mediator.
U.S. Patent No. 5,682,884 (1997, Hill et al.) discloses a strip electrode
with screen printing. The strip has an elongated support which includes a first
and second conductor each extending along the support. An active electrode,
10 positioned to contact the liquid mixture and the first conductor, has a deposit of
an enzyme capable of catalyzing a reaction and an electron mediator. A
reference electrode is positioned to contact the mixture and the second
conductor.
U.S. Patent No. 5,759,364 (1998, Charlton et al.) discloses an
15 electrochemical biosensor having an insulating base plate bearing an electrode
on its surface which reacts with an analyte to produce mobile electrons. The
base plate is mated with a lid of deformable material which has a concave area
surrounded by a flat surface so that when mated to the base plate there is formed
a capillary space into which a fluid test sample can be drawn. The side of the lid
20 facing the base is coated with a polymeric material which serves to bond the lid to
the base plate and to increase the hydrophilic nature of the capillary space.
U.S. Patent No. 5,762,770 (1998, Pritchard et al.) discloses an
electrochemical biosensor test strip that has a minimum volume blood sample
requirement of about 9 microliters. The test strip has a working and counter
25 electrodes that are substantially the same size and made of the same electrically
conducting material placed on a first insulating substrate. Overlaying the
electrodes is a second insulating substrate which includes a cutout portion that
forms a reagent well. The cutout portion exposes a smaller area of the counter
electrode than the working electrode. A reagent for analysis of an analyte
30 substantially covers the exposed areas of the working and counter electrodes in
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the reagent well. Overlaying the reagent well and affixed to the second insulating
substrate is a spreading mesh that is impregnated with a surfactant.
U.S. Patent No. 5,755,953 (1998, Henning et al.) discloses an reduced-
interference biosensor. The device generally comprises an electrode used to
electrochemically measure the concentration of an analyte of interest in a
solution. The device includes a peroxidase enzyme covalently bound to
microparticle carbon and retained in a matrix in intimate contact with the
electrode. According to this disclosure, it is the enzyme/microparticle carbon of
the device which provides a composition which is displays little sensitivity to
known interfering substances.
U.S. Patent No. 5,120,420 (1992, Nankai et al.) discloses a biosensor with
a base board having an electrode system mainly made of carbon, an insulating
layer, a reaction layer containing an enzyme layer thereon, a spacer and a cover .
The spacer creates a channel with an inlet and an outlet for holding a sample.
However, the prior art devices suffer from various shortcomings. One of
these shortcomings is interference with biosensor readings caused by other
substances in the sample fluid which can oxidize at the same potential. Prevalent
among these are ascorbic acid, uric acid and acetaminophen. As these and other
interfering substances oxidize, the current resulting from their oxidation is added
to and indistinguishable from the current resulting from the oxidation of the blood
analyte being measured. An error therefore results in the quantification of the
blood analyte.
Another shortcoming is the interference caused by red blood cells (the
hematocrit effect). This interference tends to cause an artificially high response
rate for low hematocrit levels and, conversely, an artificially low response rate for
high hematocrit levels.
Additional shortcomings of the prior art devices are that they have a more
limited linear range and require a relatively large quantity of sample volume.
Further, they require a relatively longer waiting time for development of a steady-
state response before a reading can be achieved. Each of these shortcomings
may, either individually or when combined with one or more of the other
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shortcomings, contribute to erroneous measurement readings during analysis.
Preliminary tests performed by the inventors of the present invention have shown
that the prior art which claims to reduce the effect of hematocrit on glucose
readings, were limited to and worked only in lower glucose concentrations.
5 Because of the importance of obtaining accurate glucose readings, it
would be highly desirable to develop a reliable and user-friendly electrochemical
sensor which does not have all of the drawbacks mentioned above. Therefore
what is needed is an electrochemical sensor that incorporates an interference-
correcting electrode to minimize the interference caused by oxidizable
10 substances present in the sample fluid. What is further needed is an
electrochemical sensor whose response is substantially independent of the
hematocrit of the sample fluid. What is still further needed is an electrochemical
sensor which requires less sample volume than previously required by the prior
art. Yet, what is still further needed is an electrochemical sensor which has a
15 wide linear measurement range; that is, a sensor having a reduced or negligible
interference effect and useable over a wider glucose concentration.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide an improved
20 electrochemical sensor which combines an enzyme and a mediator. It is a further
object of the present invention to provide an electrochemical sensor that
incorporates an interference-correcting electrode to minimize the interference
caused by oxidizable substances present in the sample fluid. It is a further object
of the present invention to provide an electrochemical sensor whose response is
25 substantially independent of the hematocrit levels of the sample fluid. It is still
another object of the present invention to provide an electrochemical sensor
which requires less sample volume than previously required by the prior art. It is
yet another object of the present invention to provide an electrochemical sensor
which has a wide linear measurement range.
30 The present invention achieves these and other objectives by providing an
electrochemical sensor which requires a smaller sample size and compensates
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for interference from oxidizable species in the sample and from varying
hematocrit levels. The present invention has a laminated, elongated body having
a sample fluid channel connected between an opening on one end of the
laminated body and a vent hole spaced from the opening. Within the fluid
5 channel lies at least two working electrodes and a reference electrode. The
arrangement of the two working electrodes and the reference electrode is not
important for purposes of the results obtained from the electrochemical sensor.
The working electrodes and the reference electrode are each in electrical contact
with separate conductive conduits, respectively. The separate conductive
10 conduits terminate and are exposed for making an electrical connection to a
reading device on the end opposite the open channel end of the laminated body.
The laminated body has a base insulating layer made from a plastic
material. Several conductive conduits are delineated on the base insulating
layer. The conductive conduits may be deposited on the insulating layer by
15 screen printing, by vapor deposition, or by any method that provides for a
conductive layer which adheres to the base insulating layer. The conductive
conduits may be individually disposed on the insulating layer, or a conductive
layer may be disposed on the insulating layer followed by etching/scribing the
required number of conductive conduits. The etching process may be
20 accomplished chemically, by mechanically scribing lines in the conductive layer,
by using a laser to scribe the conductive layer into separate conductive conduits,
or by any means that will cause a break between and among the separate
conductive conduits required by the present invention. The preferred conductive
coatings are gold film or a tin oxide/gold film composition. It should be pointed
25 out that although the same electrically conducting substance (gold film or tin
oxide/gold film) after scoring is used as conducting material for both working
electrodes and the reference electrode, this material itself cannot function as a
reference electrode. To make the reference electrode work, there must be a
redox reaction (e.g., Fe(CN) 6 3 " + e -> Fe(CN) 6 4 ") at the electrically conducting
30 material when a potential is applied. Therefore, a redox mediator must be
present at the conducting material used for the reference electrode.
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On top of the base insulating layer and the conductive conduits, the
laminated body has a first middle insulating layer containing cutouts for at least
two working electrodes and a reference electrode. One of the working electrodes
and reference electrode may share the same cutout, provided that the electrode
5 material (described later) disposed in the cutout is scored to isolate the working
electrode from the reference electrode. Where three cutouts are used, each
cutout corresponds to and exposes a small portion of a single conductive conduit.
The cutouts for the working electrodes are substantially the same size. The
cutout for the reference electrode may be the same or different size as the
10 cutouts for the working electrodes. The placement of all of the cutouts are such
that they will all co-exist within the sample fluid channel described above. This
first middle insulating layer is also made of an insulating dielectric material,
preferably plastic, and may be made by die cutting the material mechanically or
with a laser and then fastening the material to the base layer. An adhesive, such
15 as a pressure-sensitive adhesive, may be used to secure the first middle
insulating layer to the base layer. Adhesion may also be accomplished by
ultrasonically bonding the first middle layer to the base layer. The first middle
insulating layer may also be made by screen printing the first middle insulating
layer over the base layer.
20 The thickness of the first middle layer must be of sufficient thickness for
loading a sufficient amount of electrode material for use as an electrochemical
sensor. Each cutout contains electrode material. The electrode material has a
redox mediator with at least one of a stabilizer, a binder, a surfactant, and a
buffer. At least one of the cutouts also contains an enzyme capable of catalyzing
25 a reaction involving a substrate for the enzyme. The redox mediator is capable of
transferring electrons between the enzyme-catalyzed reaction and the working
electrode.
The laminated body also has a second middle insulating layer on top of the
first middle layer. The second middle layer is also made of a plastic insulating
30 material and creates the sample fluid channel of the laminated body. It contains
a U-shaped cutout on one end which overlays the cutouts on the first middle layer
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with the open end corresponding to the open end of the laminated body
described earlier.
The laminated body of the present invention has a top layer with a vent
opening. The vent opening is located such that at least a portion of the vent
opening overlays the bottom of the U-shaped cutout of the second middle
insulating layer. The vent allows air within the sample fluid channel to escape as
the sample fluid enters the open end of the laminated body. The sample fluid
generally fills the sample fluid channel by capillary action. In small volume
situations, the extent of capillary action is dependent on the
hydrophobic/hydrophilic nature of the surfaces in contact with the fluid
undergoing capillary action. This is also known as the wetability of the material.
Capillary forces are enhanced by either using a hydrophilic insulating material to
form the top layer, or by coating at least a portion of one side of a hydrophobic
insulating material with a hydrophilic substance in the area of the top layer that
faces the sample fluid channel between the open end of the laminated body and
the vent opening of the top layer. It should be understood that an entire side of
the top layer may be coated with the hydrophilic substance and then bonded to
the second middle layer.
The insulating layers of the laminated body may be made from any
dielectric material. The preferred material is a plastic material. Examples of
acceptable compositions for use as the dielectric material are polyvinyl chloride,
polycarbonate, polysulfone, nylon, polyurethane, cellulose nitrate, cellulose
propionate, cellulose acetate, cellulose acetate butyrate, polyester, acrylic, and
polystyrene.
The number of cutouts in the first middle layer can be one, two and three
or more. To use only one cutout, the single cutout must expose portions of two
conductive conduits. The electrode material within the single cutout is scored in
the middle to separate it into two parts; one acting as the working electrode and
the other acting as the reference electrode. Such an arrangement allows for
testing a smaller sample volume compared to two or three cutout embodiment.
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However, this embodiment lacks the interference and hematocrit correction
features of the other embodiments.
An embodiment having two cutouts is an alternative to the single cutout
version. It has one cutout serving as the working electrode and the other one
5 serving as a reference electrode. Another embodiment of the two cutout version
combines the features of making the single cutout with that of the two cutout
version. One of the cutouts containing electrode material is scored into two parts,
one part serving as a first working electrode and the second part serving as the
reference electrode. The second cutout serves as a second working electrode.
10 Such a design is an alternative embodiment of the preferred embodiment of the
present invention. This version of the two-cutout embodiment has the
interference and hematocrit correction features but also allows for measuring an
even smaller sample volume than that of the three-cutout embodiment.
In the three-cutout embodiment, two cutouts contain material for the
15 working electrodes (W1 and W2) and one for the reference electrode (R). W2
further contains the enzyme capable of catalyzing a substrate of the enzyme.
The three electrodes are positioned and sized in such a way that the resistance
of the fluid sample could be precisely measured and the possible carry-over from
W2 could be minimized. The possible electrode arrangements within the sample
20 fluid channel may be W1-W2-R, W1-R-W2, R-W1-W2, W2-W1-R, W2-R-W1, or
R-W2-W1 with the arrangement listed as the arrangement of electrodes would
appear from the open end of the laminated body to the vent opening. The
preferred position was found to be W1-R -W2; that is, as the sample fluid entered
the open end of the laminated body, the fluid would cover W1 first, then R, then
25 W2. The preferred position allows for the precise measurement of blood sample
resistance. This is necessary for good correlation between the resistance and
hematocrit level in the blood sample.
As mentioned earlier, oxidizable interferants such as ascorbic acid, uric
acid and acetaminophen, to name a few, cause inaccurate readings in the output
30 of an electrochemical biosensor. The present invention negates this effect by
subtracting the current response at W1 (first working electrode) from the current
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response from W2 (second working electrode) to calculate the enzyme
concentration in the sample fluid. This is achieved by maintaining the surface
area of W1 substantially equal to the surface area of W2. Also important is the
composition of the reagents disposed on W1 and W2. The reagents are
5 designed to have a minimal effect on the response of the interferences which also
contributes to the accuracy of the analyte measurement.
The hematocrit interference is reduced by using a two-step process. First,
the resistance (r-value) between W1 (first working electrode) and R (reference
electrode) is measured. The r-value is then used to estimate the hematocrit level
10 in the sample fluid. The following equation represents this relationship:
r=M(1-H) Eq.(1)
where r is resistance value measured in Ohms or Kilo-Ohms
H is hematocrit level
15 Wi lsa constant equal to 4.6 (r measured in Kilo-Ohms)
Second, the hematocrit level value is then used to mathematically correct
the enzyme concentration reading obtained from above. The following equation
represents the calculation performed using the calculated hematocrit level from
20 Eq. (1);
25
Ccorr — Cmea / (k 2 +k3C m ea+(k4+k 5 C me a)(1-H))
Eq. (2)
where Ccorr is the corrected analyte concentration
Cmea is the measured analyte concentration
k 2 is a constant equal to 1 .03
k 3 is a constant equal to -0.003
30 k4 is a constant equal to -0.1
k 5 is a constant equal to 0.0054
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H is the calculated hematocrit level from Eq. (1)
The constant values above have been determined for the preferred embodiment
of the present invention. Varying the surface area of the electrode areas and the
5 formulations of the reagents may require one skilled in the art to calculate new
values for constants k r k 5 in order to more accurately determine corrected
glucose concentration.
All of the advantages of the present invention will be made clearer upon
review of the detailed description, drawings and appended claims.
10
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1 is a perspective view of the present invention showing the open end,
the vent and the electrical contact points of the laminated body.
15 FIGURE 2 is an exploded, perspective view of the present invention showing the
various layers of the laminated body.
FIGURES 3A, 3B, 3C, and 3D are top views of a strip of each layer of the present
invention showing the patterns for making multiple sensors of the present
20 invention.
FIGURE 3E is a top view of a segment of the laminated strip of the present
invention showing the patterns for making multiple sensors of the present
invention.
25
FIGURES 4A and 4B are graphs showing the effect of hematocrit on the
concentration response of the present invention in normal and high
concentrations of blood glucose.
30 FIGURE 5 is a correlation of sample volume on the concentration response of the
present invention.
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FIGURE 6 is a correlation curve of the concentration readings using sensors of
the present invention versus the concentration readings of obtained on the same
samples using a YSI glucose analyzer
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The preferred embodiment of the present invention is illustrated in
FIGURES 1-6 . Figure 1 shows a sensor 10 of the present invention. Sensor 10
has a laminated body 100, a fluid sampling end 110, an electrical contact end
120, and a vent opening 52. Fluid sampling end 110 includes a sample fluid
channel 112 between a sampling end aperture 114 and vent opening 52.
Electrical contact end 120 has at least three discreet conductive contacts 122,
124 and 126.
Referring now to Figure 2, laminated body 100 is composed of a base
insulating layer 20, a first middle layer 30, a second middle layer 40, and a top
layer 50. All layers are made of a dielectric material, preferably plastic.
Examples of a preferred dielectric material are polyvinyl chloride, polycarbonate,
polysulfone, nylon, polyurethane, cellulose nitrate, cellulose propionate, cellulose
acetate, cellulose acetate butyrate, polyester, acrylic and polystyrene. Base
insulating layer 20 has a conductive layer 21 on which is delineated a first
conductive conduit 22, a second conductive conduit 24 and a third conductive
conduit 26. Conductive conduits 22, 24 and 26 may be formed by scribing or
scoring the conductive layer 21 as illustrated in Fig. 2 or by silk-screening the
conductive conduits 22, 24 and 26 onto base layer 20. Scribing or scoring of
conductive layer 21 may be done by mechanically scribing the conductive layer
21 sufficiently to create the three independent conductive conduits 22, 24 and 26.
The preferred scribing or scoring method of the present invention is done by
using a carbon dioxide (C0 2 ) laser, a YAG laser or an eximer laser. An additional
scoring line 28 (enlarged and not to scale; for illustrative purposes only) may be
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made, but is not necessary to the functionality of sensor 10, along the outer edge
of base layer 20 in order to avoid potential static problems which could give rise
to a noisy signal. Conductive layer 21 may be made of any electrically
conductive material, preferably gold or tin oxide/gold. A useable material for
5 base layer 20 is a tin oxide/gold polyester film (Cat. No. FM-1) or a gold polyester
film (Cat. No. FM-2) sold by Courtaulds Performance Films, Canoga Park,
California.
First middle layer 30 has a first electrode cutout 32 which exposes a
portion of first conductive conduit 22, a second electrode cutout 34 which
10 exposes a portion of second conductive conduit 24 and a third electrode cutout
36 which exposes a portion of third conductive conduit 26. First layer 30 is made
of a plastic material, preferably a medical grade one-sided tape available from
Adhesive Research, Inc., of Glen Rock, Pennsylvania. Acceptable thickness of
the tape for use in the present invention are in the range of about 0.003 in. (0.76
15 mm) to about 0.005 in. (0.127 mm). One such tape, Arcare® 7815, was preferred
because of its ease of handling and it showed good performance in terms of its
ability to hold a sufficient quantity of chemical reagents and to promote a
favorable blood flood speed (capillary action) through sample fluid channel 112 of
sensor 10. It should be understood that the use of a tape is not required. A
20 plastic insulating layer may be coated with a pressure sensitive adhesive, or may
be ultrasonically-bonded to base layer 20, or may be silk-screened onto base
layer 20 to achieve the same results as using the polyester tape mentioned.
The three cutouts 32, 34 and 36 define electrode areas W1, R and W2,
respectively, and hold chemical reagents forming two working electrodes and one
25 reference electrode. Typically, electrode area R must be loaded with a redox
reagent or mediator to make the reference electrode function. If R is not loaded
with a redox reagent or mediator, working electrodes W1 and W2 will not work.
Electrode areas W1 and R are loaded preferably with the same chemical reagent
to facilitate the resistance measurement described earlier. The reagents
30 preferably contain an oxidized form of a redox mediator, a stabilizer, a binder, a
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surfactant, and a buffer. Typically, the redox mediator may be at least one of
ferrocene, potassium ferricyanide and other ferrocene derivatives. The preferred
stabilizer is polyethylene glycol, the preferred binder is methyl cellulose, the
preferred surfactant is t-octyiphenoxypolyethoxyethanol, and the preferred buffer
5 is a citrate buffer. Electrode area W2 is preferably loaded with the same
chemical reagents loaded into electrode areas W1 and R but with the addition of
an enzyme capable of catalyzing a reaction involving a substrate for the enzyme
or a substrate catalytically reactive with an enzyme and a mediator capable of
transferring electrons transferred between the enzyme-catalyzed reaction and the
10 working electrode to create a current representative of the activity of the enzyme
or substrate and representative of the compound.
The cutouts and electrode areas of first layer 30 are positioned relative to-
each other and to the flow of the sample fluid in sample fluid channel 112 such
that the resistance of the sample fluid may be precisely measured and the
15 possible carryover from electrode area W2 to electrode area W1 could be
minimized. Using fluid sample end 110 of sensor 10 as a reference point, the
arrangements of the electrode areas could be W1-W2-R, W1-R-W2, R-W1-W2,
W2-W1 -R, W2-R-W1 , or R-W2-W1 . The preferred position was found to be W1 -
R-W2.
20 Second middle layer 40 has a U-shaped channel cutout 42 located at
second layer sensor end 41. The length of channel cutout 42 is such that when
second middle layer 40 is layered on top of first middle layer 30, electrode areas
W1, W2 and R are within the space defined by channel cutout 42. The thickness
of second middle layer 40 was found to be critical for the speed of the sample
25 fluid flow into sample fluid channel 112, which is filled by capillary action of the
sample fluid.
Top layer 50, which is placed over second middle layer 40, has a vent
opening 52 spaced from fluid sample end 110 of sensor 10 to insure that sample
fluid in fluid channel 112 will completely cover electrode areas W1, W2 and R.
30 Vent opening 52 is placed in top layer 50 so that it will align somewhat with the
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bottom of channel cutout 42 of second middle layer 40. Preferably, vent opening
52 will expose a portion of and partially overlay the bottom of the U-shaped cutout
42 of second middle layer 40.
5 Preparation of Reagents 1 & 2
Reagents 1 and 2 comprise the oxidized form of a redox mediator, a
stabilizer, a binder, a surfactant, and a buffer. Reagent 2, in addition, contains an
enzyme. The oxidized form of the redox mediator, potassium ferricyanide, was
found to be stable in the matrices. The quantity used in the formulation must be
10 sufficient to attain a workable linear range. The enzyme must also have sufficient
activity, purity and stability. A commercially available glucose oxidase may be
obtained from Biozyme, San Diego, California as Cat. No. G03A, about 270U/mg.
The stabilizer must be sufficiently water-soluble and be capable of stabilizing
both the mediator and the enzyme. The binder should also be capable of binding
15 all other chemicals in the reagents in electrode areas W1 , W2 and R to the
conductive surface/layer 21 of base layer 20. The preferred stabilizer is
polyethylene glycol (Cat. No. P4338, Sigma Chemicals, St. Louis, MO). The
preferred binder is Methocel 60 HG (Cat. No. 64655, Fluka Chemical, Milwaukee,
Wl). The buffer solution must have sufficient buffer capacity and pH value to
20 optimize the enzyme reaction. A 0.05M citrate buffer is preferred. The surfactant
is necessary to facilitate dispensing of Reagents 1 and 2 into cutouts 32, 34 and
36 of middle layer 30 as well as for quickly dissolving the dry chemical reagents.
The amount and type of surfactant is selected to assure the previously mentioned
functions and to avoid a denaturing effect on the enzyme. The preferred
25 surfactant is Triton X-100. The reagents are prepared as follows:
Reagent 1
Step 1: Prepare 50 mM citrate buffer (pH 5.7) by dissolving 0.1512 grams citric
acid and 1.2580 grams sodium citrate in 100 ml of deionized water.
30 Step 2: Prepare a 1% methocel 60HG solution by stirring 1 gram of methocel in
100 ml of citrate buffer from Step 1 for 12 hours.
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Step 3: Add 0.3 ml of 10% Triton X-100 into the methocel solution.
Step 4: Add 2.5 grams of polyethylene glycol into the solution from Step 3.
Step 5: While stirring, add 1 gram of potassium ferricyanide to the solution from
Step 4.
Reagent 2
Step 1-Step 4: same steps as Reagent 1 .
Step 5: While stirring, add 6.5 grams potassium ferricyanide to the solution of
Step 4.
Step 6: Add 1 .0 gram of glucose oxidase to the solution of Step 5 and stir for 1 0
minutes or until all solid materials are completely dissolved.
Electrode Construction
A piece of a gold or tin oxide/gold polyester film available from Courtaulds
Performance Films is cut to shape, as illustrated in Fig. 2, forming base layer 20
of sensor 10. A C0 2 laser was used to score the gold or tin oxide/gold polyester
film. As illustrated in Fig. 2, the film was scored by the laser such that three
electrodes at sample fluid end 110 and three contact points 122, 124 and 126
were formed at electrical contact end 120. The scoring line is very thin but
sufficient to create three separate electrical conductors. A scoring line 28 can be
made, but is not necessary, along the outer edge of base layer 20 to avoid
potential static problems which could cause a noisy signal from the finished
sensor 10.
A piece of one-sided adhesive tape is then cut to size and shape forming
first middle layer 30 so that it will cover a majority of the conductive layer 21 of
base layer 20 except for exposing a small electrical contact area illustrated in Fig.
1. Three rectangular, square or circular cutouts 32, 34 and 36 of substantially
equal size are punched by C0 2 laser (25W laser available from Synrad, Inc., San
Diego, CA). Cutouts 32, 34 and 36 define the electrode areas W1 , W2 and R
which hold chemical reagents. The size of the cutouts is preferred to be made as
small as possible in order to make the fluid sample channel 1 12 of sensor 10 as
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WO 00/73785 PCT/US00/15413
short as possible while still being capable of holding sufficient chemical reagent
to function properly. The preferred hole size for the present invention has a
typical dimension of about 0.033 in. (0.84 mm) by about 0.043 in. (1 .09 mm). As
illustrated in Fig. 2, cutouts 32, 34 and 36 are aligned with each other and having
5 a spacing of about 0.028 in. (0.71 mm) between them. The rectangular cutouts
are for illustrative purposes only. It should be understood that the shape of the
cutouts is not critical provided that the size of the cutouts is big enough to hold
sufficient chemical reagents for the electrodes to function properly but small
enough to allow for a reasonably small sample channel. As noted earlier,
10 changing the shape of the cutouts or the surface area of the cutouts may require
changing the constant values k^ks for Eq. 1 and Eq. 2. As stated previously, the
preferred arrangement of the electrodes formed in cutouts 32, 34 and 36 is W1
(working electrode 1), R (reference electrode) and W2 (working electrode 2).
0.4 microliters of Reagent 1 is dispensed into electrode areas W1 and R.
15 Reagent 1 is a mixture of a redox mediator, a stabilizer, a binder, a surfactant,
and a buffer. The preferred mixture for Reagent 1 is made by mixing the
following components in the described percentages (W/W%): about 1%
potassium ferricyanide, about 2.5% polyethylene glycol, about 1% methocel 60
HG, about 0.03% Triton X-100 and about 0.05M citrate buffer (pH 5.7). 0.4
20 microliters of Reagent 2 is dispensed into electrode area W2. Reagent 2 is a
mixture similar to that of Reagent 1 but with the addition of an enzyme capable of
catalyzing a reaction involving a substrate of the enzyme. The preferred enzyme
is glucose oxidase. The preferred mixture for Reagent 2 is made by mixing the
following percentages (WAA/%) of the following ingredients: about 6.5%
25 potassium ferricyanide, about 2.5% polyethylene glycol, about 1% methocel 60
HG, about 0.03% Triton X-100, about 0.05M citrate buffer (pH 5.7), and about 1%
glucose oxidase. After the addition of the reagents, the device was dried for
about 2 minutes at 55°C in an oven. After drying, a piece of double-sided tape
available from Adhesive Research was fashioned into second middle layer 40
30 with U-shaped channel 42. Second middle layer 40 is then layered onto first
middle layer 30. As mentioned earlier, this second middle layer 40 serves as a
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WO 00/73785 PCT/USOO/15413
spacer and defines the size of the fluid sample channel 112. Its width and length
is optimized to provide for a relatively quick moving fluid sample. The preferred
size of U-shaped channel 42 is about 0.063 in. (1.60 mm) wide by about 0.248 in.
(6.30 mm) long.
5 A piece of a transparency film (Cat. No. PP2200 or PP2500 available from
3M) is fashioned into top layer 50. A rectangular vent hole 52 is made using the
C0 2 laser previously mentioned. The preferred size of vent hole 42 is about
0.075 in. (1.91 mm) by about 0.059 in. (1.50 mm). Vent hole 52 is located
approximately 0.130 in. (3.3 mm) from fluid end 110 of sensor 10. Top layer 50 is
10 aligned and layered onto second middle layer 40 to complete the assembly, as
illustrated in Fig. 1 , of sensor 10.
Although the description of electrode construction above describes
construction for a single sensor, the design and materials used are ideal for
making multiple sensors from one piece of each layer material as shown in Fig.
15 3A-3E. This would be accomplished by starting with a relative large piece of
base layer 20 having conducting layer 21 thereon. A plurality of scored lines are
made into conductive layer 21 such that a repetitive pattern, as illustrated in Fig.
. 3A, is created using the preferred scribing method described previously whereby
each pattern will eventually define the three conductive paths 22, 24 and 26 for
20 each sensor. Similarly, a large piece of first middle layer 30, which is illustrated
in Fig. 3B and which also has a plurality of cutouts 32, 34, and 36 in a repetitive
pattern, is sized to fit over base layer 20 in such a way that a plurality of sensors
10 will be had when completed. The size of each cutout and the electrode
material disposed in the plurality of electrode areas W1, R and W2 are similar to
25 that disclosed above. After disposing Reagents 1 & 2 in their respective cutouts
and dried, a large piece of second middle layer 40 having a plurality of elongated
cutouts 42 and illustrated in Fig. 3C is layered onto first middle layer 30 such that
each elongated cutout 42 of second middle layer 40 contains corresponding
cutouts 32, 34 and 36 of first middle layer 30. A comparably-sized top layer 50
30 having a plurality of vent openings 52 in a repetitive pattern, as shown in Fig. 3D,
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is layered onto second middle layer 40. Fig. 3E is a top view of the combined
layers. The laminated strip created by the four layers 20, 30, 40 and 50 has a
plurality of sensors 10 that can be cut from the laminated strip. The laminated
strip is cut longitudinally along line A-A' at fluid sampling end 210 to form a
5 plurality of sampling apertures 114 and longitudinally along line B-B' at electrical
contact end 220 to form a plurality of conductive contacts 122, 124 and 126. The
laminated strip is also cut at predetermined intervals along line C-C forming a
plurality of individual sensors 10. Shaping of the fluid sampling end 120 of each
sensor 10, as illustrated in Fig. 1, may be performed if desired. It should be
10 understood by those skilled in the art that the order in which the laminated strip
can be cut is not important. For instance, the laminated strip may be cut at the
predetermined intervals (C-C) and then the cuts along A-A 1 and B-B' can be
made to complete the process.
The following examples illustrate the unique features of the present
15 invention which includes the compensation for varying hemotacrit levels by
measuring sample fluid resistance and nullification of the interference effects of
oxidizable species present in the sample fluid. All sensors of the present
invention were tested on a breadboard glucose meter manufactured by Nova
Biomedical Corporation of Waltham, MA. A potential of 0.35 Volts was applied
20 across the working electrodes and the reference electrode and the resultant
current signals were converted to glucose concentrations in accordance with the
disclosure of the present invention. The readings were compared to readings
(control readings) obtained on the same samples using YSI Glucose Analyzer
(Model 2300) available from Yellow Springs Instruments, Inc., Yellow Springs,
25 OH.
Example 1
Demonstration of Hematocrit Compensation
The unique design of the present invention makes it possible to measure
30 the resistance of the fluid sample. This is achieved by applying the same
reagent, Reagent 1 , to the reference electrode R and the first working electrode
. 19
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W1. The chemical reagents used in Reagent 1 are critical for accurate
measurement of the resistance. Reagent 1 can not contain a large amount of
salts or any glucose oxidase. Otherwise, the resulting resistance would not be
accurate and would be glucose dependent. For proper functioning of the present
invention, it should be noted that a minimum amount of a mediator such as
potassium ferricyanide for the reference electrode is essential.
Resistance of a sample fluid, in this case blood samples, between W1 and
r is measured at any time, preferably 20 seconds after a reading device (Nova
glucose meter) is triggered by the blood samples. Blood samples with different
hematocrit levels were prepared by spinning a whole blood sample and
recombining plasma and red blood cells in varying ratios. Hematocrit levels were
measured with a micro hematocrit centrifuge. Concentrations of glucose in the
various samples were measured by sensors of the present invention (Cmea) and
by a YSI blood glucose analyzer (the control), Model 2300, Yellow Springs
Instruments, Inc., Yellow Springs, OH. Equations (1) and (2), previously
mentioned, were used to calculate the corrected glucose concentration (C CO fr)
measured by sensors of the present invention to demonstrate the hemotacrit
compensation feature of the present invention. The data obtained was plotted
and Figs. 4A and 4B show two graphs representing the percent correlation of the
readings obtained using sensors of the present invention with the Nova glucose
meter to the readings obtained for the samples using the YSI blood glucose
analyzer at low and high levels of glucose in samples with varying hematocrit
levels.
Example 2
Demonstration of Interference Free Feature
The unique design of the present invention makes it possible to eliminate
interference from oxidizable substances such as ascorbic acid, acetaminophen,
uric acid, and other possible interferants present in the sample. This is achieved
by subtracting the response obtained from W1 from the response obtained at W2,
and is represented by the following equation:
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I = Iw*-Iwi Eq. (3)
where Iw 2 is the current at W2 (second working electrode)
5 Iwi is the current at W1 (first working electrode)
I is the difference between W2 and W1 and represents the
current due to oxidation of the mediator of its reduced
form, which is proportional to the glucose
concentration in the sample
10
Because W1 and W2 have the same surface area, the potential
interference present in the sample fluid should give relatively identical signals
from each working electrode. Even though W1 and W2 had different reagents, it
was found that there was no remarkable difference in the response to the
15 interference. Thus, the difference in current response obtained in blood samples
was due to the glucose present in the samples. This was tested by spiking
normal and high glucose blood samples with 1 mM and 5 mM ascorbic acid,
acetaminophen and uric acid. Table 1 shows the percentage response change of
the readings obtained with sensors of the present invention and various
20 commercially available sensors (referred to as Strip 1 , Strip 2, Strip 3, and Strip
4) in blood samples having a concentration of 100 mg/dL glucose and 300 mg/dL
upon addition of the interferents.
Table 1 - Response change (%) Upon Addition of interferent
100 mg/dL glucose : ascorbic acid
mM
Nova
Strip 1
Strip 2
Strip 3
Strip 4
0
0
0
0
0
0
1
4
26.7
19.8
21.3
6
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5
4.5
133.6
115.8
151.5
Error
300 mg/dL glucose : ascorbic acid
0
0
0
0
0
0
1
-0.9
7.2
14
14.7
-4.3
5
-0.5
Hi
107.3
Hi
Error
100 mg/dL glucose : acetaminophen
0
0
0
0
0
0
1
3.0
34.7
5.7
50
-8.2
5
3.4
90.8
38
136
-13.4
300 mg/dL glucose : acetaminophen
0
0
0
0
0
0
1
-3.4
20
3.5
18
-7.2
5
-3.5
39
8.0
23.7
-17.5
100 mg/dL glucose : uric acid
0
0
0
0
0
0
1
-2.7
32
35
35
-1.3
300 mg/dL g
lucose : uric acid
0
0
0
0
0
0
1
-4.5
10
15
7
-1
From the test data, one observes that the readings obtained from sensors
of the present invention show essentially no change in the presence of 1 mM and
5 mM ascorbic acid and acetaminophen, and 1 mM uric acid. All commercially
5 available sensors except one, Strip 4, suffer from serious interference. Strip 4
showed an "error" for 5 mM ascorbic acid. At concentrations of 300 mg/dL
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glucose, sensors of the present invention also showed no interference (response
change of less than 5%) upon spiking the samples with 1 mM and 5 mM ascorbic
acid. The commercially available sensors showed about 7% to about 15%
response increase for 1 mM ascorbic acid spiked samples, and showed a "Hi"
5 reading for 5 mM ascorbic acid spiked samples. Strip 4 again showed an "error"
for 5 mM ascorbic acid spiked samples. In samples containing acetaminophen
and uric acid, all commercially available strips showed varying degrees of error
except for Strip 4 in samples containing uric acid.
10 Example 3
Demonstration of Minimum Sample Volumes Feature
The unique design of the present invention enables the measurement of
sample sizes smaller than which have heretofore been possible. Blood samples
are applied to the sensors and the samples travel along the fluid sample channel
15 to the venting hole. The blood volume required for measurement of blood
glucose is determined by the channel volume. The calculated volume for the
present invention is 1 .44 microliters. In order to test the volume effect on sensor
response, different blood sample volumes were applied to the sensors and the
resulting concentration readings were plotted against volume. The test data is
20 shown in Fig. 5.
Sensors of the present invention show no dependence of the response on
. the sample volume if the volume is above 1 .5 microliters. It was found that
sensors of the present invention still gave reasonable readings on sample sizes
as low as 1.0 microliters. This is possible because the hydrophilic character of
25 Reagent 1 applied to W1 and R, and Reagent 2 applied to W2 permitted the
sample to cover the electrode areas even though the blood volume did not fill the
entire sample channel.
Example 4
30 Demonstration of Wide Linear Range and Precision Feature
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A sample of venous blood was collected and separated into several
aliquots. Each aliquot was spiked with different glucose concentrations ranging
from 35 to 1000 mg/dL The aliquots were each measured with a YSI glucose
analyzer and then with sensors of the present invention using the Nova glucose
5 meter. Sensors of the present invention show a linear relationship of current
response vs. glucose concentration from 35 to 1000 mg/dL. The concentration
readings were plotted against the concentration values obtained using the YSI
meter (the control) and are illustrated in Fig. 6.
A regression coefficient of 0.9988 indicated a near perfect match with the
10 readings obtained with the YSI blood glucose analyzer. The same aliquots were
tested using four different commercially-available sensors with their
accompanying meters. The commercially-available sensors showed a linear
• response only up to about 600 mg/dL. Above the 500-600 mg/dL range, all
commercially available sensors displayed "Hi" as the test result.
15 The precision of the sensors of the present invention was investigated at
the same glucose level range from about 35 to 1000 mg/dL. Four different
batches of sensors of the present invention were used in the precision tests.
Typically, the relative standard deviation was about 9.5%, 5.0%, 3.5%, 2.9%, and
2.6% for samples containing 35, 100, 200, 500, and 1000 mg/dL levels of
20 glucose, respectively.
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What is claimed is:
1. A disposable electrode strip for testing a fluid sample comprising:
a laminated strip having a first strip end, a second strip end and a vent
opening spaced from said first strip end, said laminated strip
comprising a base layer with at least three electrodes delineated
thereon, a reagent holding layer carried on said base layer, said
reagent holding layer having at least two cutouts, a channel forming
layer carried on said reagent holding layer, and a cover;
an enclosed channel between said first strip end and said vent opening, said
enclosed channel containing said at least two cutouts;
a first reagent disposed in a first cutout of said at least two cutouts forming a
reference electrode;.
a second reagent disposed in a second cutout of said at least two cutouts
forming a first working electrode, said second reagent being
substantially similar to said first reagent and containing an enzyme;
and
conductive contacts at said second strip end and insulated from said
enclosed channel.
2. The electrode strip of Claim 1 further comprising:
a third cutout in said reagent holding layer and contained within said
enclosed channel; and
a third reagent disposed in said third cutout forming a second working
electrode, said third reagent being substantially similar to said first
reagent.
3. The electrode strip of Claim 2 wherein said first reagent, said second
reagent and said third reagent contain a redox mediator.
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4. The electrode strip of Claim 3 wherein said redox mediator is at least one
metal complex.
5. The electrode strip of Claim 3 wherein said at least one redox mediator is
5 potassium ferricyanide and other inorganic and organic redox mediators.
6. The electrode strip of Claim 1 wherein said base layer has a conductive
coating disposed thereon for forming said at least two electrodes.
10 7. The electrode strip of Claim 6 wherein said conductive coating is gold.
8. The electrode strip of Claim 6 wherein said conductive coating comprising
gold and tin oxide.
15 9. The electrode strip of Claim 6 wherein said base layer, said reagent holding
layer, said channel forming layer, and said cover are made of a plastic
dielectric material.
10. The electrode strip of Claim 9 wherein said plastic material is selected from
20 the group consisting of polyvinyl chloride, polycarbonate, polysulfone, nylon,
polyurethane, cellulose nitrate, cellulose propionate, cellulose acetate,
cellulose acetate butyrate, polyester, acrylic, and polystyrene.
11. The electrode strip of Claim 1 wherein said enclosed channel is hydrophilic.
12. The electrode strip of Claim 1 wherein said enclosed channel has a volume
of about 1 .44 microliters.
13. The electrode strip of Claim 1 wherein said cover has a hydrophilic coating
30 on at least one side.
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14. The electrode strip of Claim 3 wherein said first reagent, said second
reagent and said third reagent further contain at least one of a stabilizer, a
binder, a surfactant, and a buffer,
5 15. The electrode strip of Claim 14 wherein said stabilizer is a polyalkylene
glycol, said binder is a cellulose material, and said surfactant is a
polyoxyethylene ether.
16. The electrode strip of Claim 15 wherein said stabilizer is polyethylene
10 glycol, said binder is methyl cellulose, said surfactant is t-
octylphenoxypolyethoxyethanol, and said buffer is a citrate buffer.
17. The electrode strip of Claim 14 wherein said first reagent, said second
reagent and said third reagent are made from a mixture having starting
15 components comprising about 1wt% to about 6.5wt% of said redox
mediator, about 2.5wt% of said stabilizer, about 1wt% of said binder, and
about .03wt% of said surfactant in said buffer.
18. The electrode strip of Claim 17 wherein said citrate buffer is about 0.05M.
20
19. The electrode strip of Claim 1 wherein said channel forming layer has a
thickness sufficient to optimize the flow of said fluid sample along said open
path.
25 20. The electrode strip of Claim 1 9 wherein said thickness is about 0.007 inches
(0.1778 mm).
21. The electrode strip of Claim 16 wherein said first reagent and said second
reagent are made of a mixture having starting components comprising about
30 1 wt% of said potassium ferricyanide, about 2.5wt% of said polyethylene
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glycol, about 1 wt% of said methyl cellulose, about .03wt% of said t-
octylphenoxypolyethoxyethanol, and said citrate buffer is about 0.05M.
22. The electrode strip of Claim 16 wherein said third reagent is made of a
5 mixture having starting components comprising about 6.5wt% of said
potassium ferricyanide, about 2.5wt% of said polyethylene glycol, about
1wt% of said methyl cellulose, about .03wt% of said t-
octylphenoxypolyethoxyethanol, and said pH buffer is about a 0.05M citrate
buffer, and about 1wt% of said enzyme.
10
23. The electrode strip of Claim 22 wherein said enzyme is glucose oxidase.
24. The electrode strip of Claim 2 wherein the surface area of said first working
electrode is substantially same as the surface area of said second working
15 electrode.
25. A method of using an electrode strip for determining the concentration of an
analyte, said electrode strip having a first working electrode, a second
working electrode and a reference electrode wherein said first working
20 electrode contains an enzyme capable of catalyzing a reaction involving a
substrate for the enzyme, said first working electrode, said second working
electrode and said reference electrode being disposed in a fluid sample
channel for measuring a fluid sample, said method comprising:
disposing said fluid sample into said channel of said electrode strip;
25 applying a potential between said reference electrode and said first working
electrode which contains said enzyme;
measuring a first current generated between said first working electrode and
said reference electrode and correlating said first current to a
concentration of said analyte in said fluid sample;
28
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WO 00/73785 PCT/US00/15413
measuring a resistance value of said fluid sample between said second
working electrode and said reference electrode;
applying said resistance value to a first equation and determining the
hematocrit level of said fluid sample; and
5 calculating a corrected concentration of said analyte using a second
equation to correct for the presence of hematocrit in said sample.
26. The method of Claim 25 wherein said method further comprising:
applying a potential between said reference electrode and said second
10 working electrode;
measuring a second current generated between said second working
electrode and said reference electrode;
subtracting said second current from said first current and obtaining a
current difference, correlating said current difference to a concentration
15 of said analyte in said fluid sample.
27. The method of Claim 26 wherein said method further includes triggering
said current measuring step when said fluid sample contacts said first
working electrode, said second working electrode and said reference
20 electrode creating said first current and said second current.
28. The method of Claim 27 wherein said method further includes reading a
current value for each of said first current and said second current at about
a time where said current values for each of said first current and said
second current reach a steady-state.
25 29. The method of Claim 28 wherein said reading is taken at about 20 seconds
after said current measuring step is triggered.
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30. A disposable electrode strip for detecting or measuring the concentration of
at least one analyte in a fluid sample, said electrode strip comprising:
an insulating base strip having a first base end and a second base end;
a conductive layer disposed on one side of said base strip and scribed to
5 delineate at least three electrically-distinct conductive paths;
a first electrical insulator sized smaller than said insulating base strip and
overlaying a substantial portion of said conductive layer, said first
insulator having at least a first cutout portion and a second cutout
portion spaced from said first base end, said first cutout portion
10 exposing a limited area of a first of said at least three conductive paths
and said second cutout portion exposing a limited area of a second and
a third of said at least three conductive paths;
at least two electrode materials wherein a first material of said at least two
electrode materials is a reagent for measuring the concentration of said
15 at least one analyte and wherein a second material of said at least two
electrode materials is a material suitable for use as a reference
material and for measuring the resistance of said fluid sample, said first
material being disposed in said first cutout portion and said second
material being disposed in said second cutout portion, said second
20 material being scored to isolate said second material disposed on said
second of said at least three conductive paths from said second
material disposed on said third of said at least three conductive paths;
a second electrical insulator sized to fit over and coextensive with said first
electrical insulator, said second insulator having an opening configured
25 to expose an area of said first insulator a limited distance from said first
base end of said insulating base strip, said area including said at least
two cutout portions of said first insulator; and
a third electrical insulator sized to fit over and coextensive with said second
insulator creating a sample fluid channel, said third insulator having a
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WO 00/73785 PCT/US00/15413
third insulator vent aperture spaced from said first base end and
configured to expose at least a small portion of said opening of said
second insulator.
5 31. The strip of Claim 30 wherein said sample fluid channel has a volume of
1.44 microliters.
32. The strip of Claim 30 wherein said sample fluid channel is hydrophilic.
10 33. The device of Claim 30 wherein said first material and said second material
are mixtures having starting components comprising a redox mediator, a
stabilizer, a binder, a surfactant, and a buffer.
34. The strip of Claim 33 wherein said redox mediator is at least one metal
1 5 complex selected from the group consisting of ferrocene, ferrocene
derivatives and potassium ferricyanide, said stabilizer is a polyalkylene
glycol, said binder is a cellulose material, said surfactant is a
polyoxyethylene ether, and said buffer has a pH of about 5 to about 6.
35. The strip of Claim 34 wherein said mediator is potassium ferricyanide, said
20 stabilizer is polyethylene glycol, said binder is methyl cellulose, said
surfactant is t-octylphenoxypolyethoxyethanol, and said buffer is a citrate
buffer.
36. The strip of Claim 35 wherein said first reagent is made of a mixture having
starting components comprising about 1 wt% of said potassium ferricyanide,
25 about 2.5wt% of said polyethylene glycol, about 1wt% of said methyl
cellulose, and about 0.03wt% of said t-octylphenoxypolyethoxyethanol in
said citrate buffer.
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37. The strip of Claim 35 wherein said second reagent is made of a mixture
having starting components comprising about 6.5wt% of said potassium
ferricyanide, about 2.5wt% of said polyethylene glycol, about 1 wt% of said
methyl cellulose, about 0.03wt% of said t-octylphenoxypolyethoxyethanol,
and about 1wt% of an enzyme in said citrate buffer.
38. The strip of Claim 37 wherein said enzyme is glucose oxidase.
39. The strip of Claim 30 wherein said insulating base strip, said first electrical
insulator, said second electrical insulator, and said third electrical insulator
are made from a plastic material selected from the group consisting of
polyvinyl chloride, polycarbonate, polysulfone, nylon, polyurethane,
cellulose nitrate, cellulose propionate, cellulose acetate, cellulose acetate
butyrate, polyester, acrylic, and polystyrene.
40. A method of making multiple, disposable sensors wherein each sensor has
a first working electrode, a second working electrode and a reference
electrode, wherein said first working electrode contains an enzyme capable
of catalyzing a reaction involving a substrate for the enzyme, said first
working electrode, said second working electrode and said reference
electrode being disposed in a fluid sample channel for measuring a fluid
sample, said method comprising:
obtaining a base strip of an insulating material having a layer of conductive
material disposed thereon, said base strip having a first edge and a
second edge;
scribing in said conductive material a plurality of lines in a repetitive pattern
wherein said plurality of lines contain a repetitive pattern forming three
conductive paths in each of said repetitive pattern;
disposing a first middle layer of insulating material over said base strip, said
first middle layer having a repetitive pattern of three cutouts wherein
each cutout of each of said repetitive pattern exposes an electrode
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portion of each of said three conductive paths of each repetitive pattern
wherein said repetitive pattern of said three cutouts are spaced from
said first edge of said base strip, and wherein said first middle layer is
sized to expose a contact portion of each of said three conductive
5 paths of each repetitive pattern for a distance from said second edge of
said base strip;
disposing a first reagent material on two of said three cutouts of each
repetitive pattern and a second reagent material on the other of said
three cutouts of each repetitive pattern;
10 drying said first reagent material and said second reagent material;
overlaying a second middle layer of insulating material over and coextensive
with said first middle layer; said second middle layer having a plurality
of elongated cutout portions in a repetitive pattern wherein each of said
elongated cutout portions exposes a corresponding repetitive pattern of
15 said three cutouts said first middle layer;
disposing a top layer of insulating material over and coextensive with said
second middle layer, said top layer having a plurality of vent openings
in a repetitive pattern wherein each of said vent openings exposes a
portion of a corresponding repetitive pattern of said elongated cutout
20 portion furthest from said first edge of said base strip; and
separating each of said repetitive pattern forming one of each of said
disposable sensors.
The method of Claim 40 further comprising drying said first reagent material
and said second reagent material at a temperature and for a length of time
sufficient to allow said first reagent material and said second reagent
material to solidify and adhere to each of said electrode portion of each of
said repetitive pattern of said three conductive paths.
41.
25
33
WO 00/73785 PCT/US00/15413
42. The method of Claim 40 further comprising cutting along said first edge of
each of said sensors and transverse to said sensors a predetermined
distance creating a sample inlet port.
34
WO 00/73785
1/6
PCT/USOO/15413
Fig. 2
2/6
PCT/USOO/15413
210
20
(
26
4
220
I
24
\
22
Fig. 3 A
210
■32
□
□
□
□
□
□
"5T
□
100
□ H=P
34
36
220
30
Fig. 3 B
210
220
Fig. 3 D
WO 00/73785 PCT/US00/1S413
4/6
B
210
5
220
V
126 124122
□
P
B'
Fig. 3 E
WO 00/73785
5/6
PCT/USOO/15413
Cysi = 108 mg/dL (H=45%)
150
NOVA/YSI% 100
50
00 0.20 0.40 0.60
1 HEMATOCRIT ™ T
Fig. 4A
150
100
NOVA/YSI % 50
Cys. = 403 mg/dL (H=45%)
ft mJBL i ft— j£Lm—& i @t ■
0.00 0.20 0.40 0.60
1 HEMATOCRIT 1
Fig. 4B
WO 00/73785
6/6
PCT/US00/1S413
100
80
Response
mg/dL
60
40
20
2 3 4
Volume, (microliters)
Fig. 5
1000-
800
YSI Reading 6Q0
mg/dL
400
R = 0.9988
200
"~l
200
I
400
~1
800
600
Test Stip Reading, mg/dL
1000
Fig. 6
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